We have continued to develop selective biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), optical microscopy and spectroscopies. In collaboration with NIH intramural and extramural scientists, we are working toward broader and more insightful biomedical applications of multifunctional, multimodal, AFM imaging and single molecule force spectroscopy (SMFS) for cellular and macromolecular studies. On biomedical applications, we have continued our broad range of collaborations that include: (1) Our collaborations on multifunctional nanomedicine and theranostics with Dr. Xiaoyuan Chen (Laboratory of Molecular Imaging and Nanomedicine, NIBIB) and co-investigators. Focusing on understanding and fighting cancer and other major diseases, we have contributed to several published and ongoing studies such as using tumor-specific formation of enzyme-instructed supramolecular assemblies as cancer theranostics. We are applying Bio-AFM and QCM-D methodology to investigate nanoparticle theranostics, cancer cells and stem cells, and other biomedical systems. (2) Our commitment to developing a better clinical vaccine toward enhanced immunological response and eventual eradication of malaria. Over several years and via Bio-AFM and related bioanalysis, we have investigated the macromolecular structure and nanomechanical properties of more malaria vaccine candidates and related samples with Dr. David Narum (NIAID, NIH) and other collaborators. Bio-AFM imaging and force spectroscopy characterization at single macromolecule and assembly level are helping to define these vaccine constructs along the developmental phases. In this year, we worked on characterizing Plasmodium falciparum circumsporozoite protein (CSP) and extracted lipid rafts to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. (3) Our multi-year Bio-AFM studies of protein clathrin and assemblies with collaborators including Prof. Eileen Lafer and Prof. Rui Sousa (Univ. Texas Health Sciences Center, San Antonio), Dr. Ralph Nossal (NICHD) and Dr. Dan Sackett (NICHD) in the key area of the receptor-mediated endocytosis and intracellular trafficking. Related to exocytosis and endocytosis, we are collaborating also with Dr. Ling-gang Wu (NINDS) and coworkers to better understand synaptic transmission and neuronal communications in brain. And we expanded bio-AFM studies of microtubules interacting with several approved or developing anti-cancer drugs. (4) Finally, our other continuing and new collaborations critically important to disease mechanisms and bionanotechnology. For example, we are collaborating with Dr. Andrew Doyle (NIDCR), Dr. Kenneth Yamada (NIDCR), and Dr. Raimon Sunyer (Institute for Bioengineering of Catalonia, Spain) on nanomechanics and structural properties of reconstituted extra cellular matrix (ECM)-like collagen gels. And with Dr. Curtis Meuse (NIST) and coworkers, we have advanced our Bio-AFM and biophysical studies of amyloid-beta fibrils in the Alzheimer's disease, especially toward resolving assembly pathways in physiologically relevant fluid and surface environments.